NEW SPONTANEOUS AND HUMANIZED MODELS OF UVEITIS: (1) Birdshot chorioretinopathy (BC) is strongly associated with HLA-A29. To study the association between HLA-A29 and uveitis, we applied to and received a Bench to Bedside award for FYs 201112. As part of the project we developed HLA-A29 transgenic mice using a construct obtained from Jacques Cohen, INSERM, Paris, who cloned the HLA-A29 gene from a BC patient. The mice developed ocular pathology resembling BC within the first few months of life. Unexpectedly, this turned out to be due to the hitherto unknown presence of the rd8 mutation of the Crb1 gene expressed by the founder mouse from the C57BL/6N strain. Extensive investigation revealed that C57BL/6N mice from all major vendors (but not C57BL/6J kept by Jax) express the rd8 mutation and have the associated phenotype. This finding has grave implications for the vision research community, as some (many?) published ocular phenotypes may have been due to this mutation (Mattapallil et. al. IOVS 2012). We are currently rederiving this strain. (2) We continue to collaborate with Drs. Warren Strober (NIAID) and Guanxun Meng (Shanghai Pasteur Institute) on the study of ocular inflammation in mice with an inflammasome mutation. These mice are knock-in for a mutated NLRP3 gene associated with Muckle-Wells syndrome, which among its pathologies is also associated with ocular inflammation. Results indicate that there are effects on cytokine responses and on pathology associated with EAU and LPS induced uveitis, models in which inflammasome responses are heavily involved. (3) We have derived three lines of T cell receptor (TCR) Tg mice that express different copy numbers of a TCR specific to the major epitope of IRBP, named (from high expression to low) R161H, R161M and R161L. R161H and to a lesser extent R161M mice develop spontaneous uveitis by 2 months of age. R161L mice develop minimal or no spontaneous disease. These mice thus represent a new model that permits to study mechanisms of spontaneous uveitis and serve as a source of retinal antigen-specific T cells, permitting for the first time to study their development and function. Using these mice, insights are being gained into mechanisms of autoimmune uveitis. (Horai et al., in preparation) FUNDAMENTAL MECHANISMS IN TOLERANCE, IMMUNITY AND AUTOIMMUNITY TO RETINAL ANTIGENS (1) Interestingly, spontaneous uveitis was ameliorated in R161H mice treated with a broad-spectrum antibiotic mixture from before birth, resulting in a drastically altered gut bacterial flora. This suggests that endogenous bacterial flora contributes to development of spontaneous ocular autoimmune disease. IL-17-producing TCR Tg R161H T cells were enriched in the lamina propria of the gut of untreated (but not antibiotic-protected) mice, and we hypothesize that they are activated there by the bacterial flora and migrate to the eye to elicit uveitis. Data (Horai et al, manuscript in preparation). We are currently attempting to dissect which component(s) of the flora is(are) responsible. (2) crossing R161H mice to IFN-g deficient or IL-17 deficient mice unexpectedly revealed a major role for IFN-g in the spontaneous disease. IFN-g-/- R161H mice, but not IL-17-/- R161H mice, had severely reduced spontaneous uveitis scores. These findings raise the possibility that IL-17 produced by R161H cells in the gut, as described above, is a marker of the pathogenic cells but not necessarily the pathogenic cytokine itself. (3) Using IRBP specific T cells from R161H mice crossed to FoxP3-GFP reporter mice we re-examined the concept of local immune privilege in vitro and in vivo. Our data show that uncommitted T cells rapidly convert in ocular fluids as well as within the living eye to functional FoxP3+ Tregs in a process involving retinal antigen recognition, de novo FoxP3 induction and proliferation. The process is in part dependent on retinoic acid within the eye, which thus fulfills a dual role: in vision and in ocular immune privilege. Importantly, only nave T cells could be converted, but effector-memory retina-specific T were resistant to conversion, and instead caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. (Zhou et al, J Immunol 2011 and Zhou et al., J. Immunol 2012). (4) Our previous work showed that autoimmunity to retina could be either Th17 or Th1 driven. Because immune responses have inherent plasticity, targeting only Th1 or only Th17 could drive the response down the other pathway while continuing to fuel pathology. We therefore sought an approach that would concurrently inhibit both. IL27p28 binds to gp130 and can block both IL-6 and IL-27 signaling, which are involved in Th17 and Th1 responses, respectively. Overexpression of IL27p28 in mice leads to protection from EAU induced by either Th1 or by Th17 cells. Furthermore, similar effects of IL-27p28 in vitro are seen on human Th1 and Th17 polarized cells. This could point to IL-27 and/or its subunits as a therapeutic mechanism in immunologically complex diseases such as uveitis. (Chong et al, submitted). (5) IL-22 is a cytokine produced by Th17 cells and is present in inflammatory sites; however, its effects on the tissue are controversial. It has been reported to have both pro-inflammatory and protective effects, depending on the site and the model. We used IL-22 and IL-22-receptor deficient mice and anti-IL-22 antibodies to examine effects of IL-22 modulation on EAU. Our data suggest that IL-22 has a local anti-inflammatory and tissue-protective role in the eye (Mattapallil et al., in preparation). (6) We examined whether T regulatory (Treg) cells found in uveitic eyes are (i) IRBP specific, (ii) functionally suppressive, and (iii) may play a role in natural resolution of disease. Using IRBP-MHC dimers as well as FoxP3 reporter and FoxP3 deleter mice, we found that the T cell infiltrate in uveitic eyes of mice that have a polyclonal T cell repertoire is highly enriched in IRBP-specific Treg and Teff cells. Unlike what has been reported for Treg in other inflammatory sites, Treg in uveitic eyes are unimpaired functionally. Finally, FoxP3+ Treg appear to play a role in the natural resolution of EAU and in the maintenance of remission. (Silver et al, in preparation) EFFECTS OF INNATE IMMUNE RESPONSES ON AUTOIMMUNITY: The innate immune response directly affects immunopathogenic processes and also impacts on adaptive immunity. (1) We previously identified a population of NKT cells that express the IL-23R constitutively and produce IL-17 independently of IL-6 and IL-21 (NKT17). Recent data indicate that IL-17 production in these cells may involve a unique signaling pathway that bypasses STAT3 under some conditions. Furthermore, have identified a novel population of non-NKT innate T cells that rapidly produce high amounts of IL-17 upon T cell receptor and IL-23 receptor ligation, similarly to NKT17. These cells lack both CD4 and CD8 expression (double negative = DNT) and express the unique PLZF transcription factor characteristic of innate T cells. We are currently studying this cell population with the goal of defining its role in host defense and tissue pathology (A. Hansen, in preparation). (2) The cytokine IFN-g can be either protective or proinflammatory in autoimmunity, which is an unsolved paradox. Our recent data suggest that there is a positive feedback loop between NK cell derived IFN-g acting on DC to produce IL-27, which in turn augments the IFN-g; response. This is in line with earlier data published by our lab (Grajewski, Hansen et al., J Immunol 2008) that innate IFN-g; production dampens subsequent adaptive IFN-g and IL-17 responses and protects from EAU (Chong et al, in preparation).